Method of producing citric acid by fermentation



/NVENTOR nge-PARO l ATTORNEY MEHR/CK March 26, 1963 M. w. sHEPARD METHOD oF PRoDucING CITRIC ACID BY FERMENTATION Filed April 4, 1960 EFFECT OF NITROGEN ADDITIONS UPON RATE 0F PRODUCT/ON OF C [77E/C` AC/D United States Patent diana Filed Apr. 4, 1960, Ser. No. 19,881 7 Claims. (Cl. 195-36) 'Ihis invention relates to the production of citric acid from carbohydrate containing materials by submerged vegetative fermentation methods. More specifically, this invention relates to a method for improving the yield of citric acid in a submerged fermentation process. In one of its more particular aspects this invention relates to the nutrition of citric acid producing fungi.

Citric acid -has been produced in commercial quantities by the fermentation of carbohydrate materials using various strains of citric acid producing fungi. Certain strains of Aspergillus niger have proved to be particularly useful for this purpose, In addition, various strains of other species of fungi such as Aspergillus clavrztus, Aspergillus wentii, Aspergillus luclzuensis, Penicillzlm citrinum, Pencllium luteum, Mucor priformis and others have been used with varying degrees of success.

In such citric acid fermentations it has been found essential to provide the citric acid producing organism with certain nutrients such as nitrogen, phosphorus, potassium and magnesium, which are required for growth of the organism and for stimulation of production of citric acid.

In general a suitable fermentation medium may be prepared by addition to a fermenter of the carbohydrate v to be fermented together with the various nutrient materials. The fermentation medium or broth is then inoculated with spores of the organism being used. Fermentation is allowed to proceed under carefully controlled conditions of temperature and pH for a period of time sufficient to obtain useful yields of citric acid. In this process it is generally found that the rate of production of citric acid increases steadily with passage of time until the rate reaches a maximum after which the rate of production of citric acid begins to fall off. In a few days the rate has fallen to a point at which production is no longer economical.

It is a major object of this invention to prolong the length of time during which citric acid production proceeds at a useful and economical rate.

Another object of this invention is to provide a process for the production of citric acid which is capable of producing increased yields of citric acid.

A further object of this invention is to provide a process for production of citric acid which utilizes a larger proportion of the carbohydrate material used for fermentation.

Other objects and advantages of this invention will become apparent in the course of the following detailed disclosure and description. Y l"This invention consists broadly in the addition of Va nitrogen nutrient to the citric acid fermentation broth at 3,083,144 Patented Mar. 26, 1963 this point can be readily ascertained by analysing samples from a control run of citric acid production in which no nitrogen is added after the fermentation begins. By determining the rate of citric acid production at various times during the fermentation cycle, and plotting this rate against the age of the fermentation it is possible to determine the point of maximum rate of citric acid production and thereby the point at which the nitrogen nutrient should be added. It should be pointed out, however, that while this procedure is desirable it has been found that enhanced production of citric acid is accomplished by nitrogen additions throughout the course of fermentation starting at any time after the tirst stage of growth of the citric acid mycelia. For example, additions from the third through the ninth day of the fermentation cycle have proved effective to produce greatly improved yields of citric acid and considerably more satisfactory utilization of the carbohydrate feed. In particular, additions of nitrogen nutrient from about the third day of fermentation to about the seventh day have proved eminently satisfactory for this purpose.

It will be appreciated that the nitrogen nutrient selected for addition to the fermentation medium at this time is in addition to any nitrogen which may have been originally charged to the fermentation medium. Such initial charge, which is present before development of citric acid producing mycelia, has been found to be generally effective to promote a certain amount of growth of the mycelia. However, it is now known that this initial charge is not effective to cause production of optimum yields of citric acid, yet the mere increasing of the concentration of nitrogen originally charged does not have the desired effect either. It is only by following the practice of the present invention, namely by feeding to the fermentation medium an effective quantity of nitrogen at a stage during the fermentation at which utilization of the increased concentration of nitrogen is possible that optimum yields of citric acid and corresponding utilization of the carbohydrate medium are obtained.

The nitrogen nutrients used for the purpose of this invention are compounds which are capable of furnishing ammonium ions to the fermenting broth. For example, ammonia, both in gaseous and aqueous form, ammonium hydroxide and ammonium carbonate may be used as the nitrogen nutrient. The commercial grade of ammonium carbonate, which is satisfactory for use for this purpose, is actually a mixture of ammonium bicarbonate and f ammonium carbamate. Other compounds which serve as a time subsequent to the beginning of fermentation. More particularly, this invention concerns nitrogen addition at a point in the fermentation cycle after the development of citric acid producing mycelia.

Inaccordance with the improved process of this invention it is generally possible to increase the yield of citric acid by the addition of a suitable nitrogen source at any time subsequent to the development of citric acid producing mycelia. It is desirable to add the nitrogen nutrient ata point in the fermentation cycle near where the rate of productionv of citric acid begins to drop ott from theV `i maximum. This point may Vary from batch to batch and is, of course, dependent upon the make-up of the fermentation medium and the fermentation conditions. However,

a source of the ammonium ion may also be used in the process of this invention. Such compounds include, for example, other ammonium salts such as ammonium nitrate, ammonium sulfate and ammonium chloride, which are also capable of furnishing ammonium ions. Ammonia is preferred Vfor this purpose because of the ease of handling and convenience of use of the various forms of ammonia.

The concentration of nitrogen nutrient added during the fermentation may be varied in accordance with the composition of the fermentation medium and the conditions of fermentation so that an amount of nutrient is used Vwhich is suflicient to maintain the maximum rate of production of citric acid beyond the point at which the rate normally would fall olf. The addition of an amount of a vnitrogen nutrient sufficient to give a concentration in the fermentation broth of from about 50 parts per million of nitrogen to about 5,000 parts per million of nitrogen is generally found adequate to insure stabilization of citric -acid production at a high rate for an extended period of time. By virtue of this maintenance of high production rates it becomes possible to shorten the over-all times of fermentation and still realize economical yields of citric acid.

While it is not desired to postulate a mechanism which may be responsible for this observed increase in yield of critic acid, it is suggested that the additional source of nutrient nitrogen may result in metabolic changes in the citric acid producing organism so as to enhance its citric acid producing ability.

EXAMPLE II The fermentation broths were made up according to the procedure of Example I except that the range of It is known, for example,

sugar used was from 22,400 lb. to 25,300 lb. (16.3-

that there is a pronounced change in the morphorolgy of citric acid producing strains of Aspergillus niger after 18-470) Per batch POaSSlUm amd Phosphate Was Present about 16 hours to about 20 hours of fermentation.

in a concentration of 140 parts per million and magnesium sulfate was used at the 1,000 parts per million level. No ammonia was added after the start of fermentation. defence The rates of citric acid production (grams of citric acid The drawing illustrates the effect of nitrogen additions produced per 100 ml. per day) for ten runs were averaged and the averages were plotted against the age of the fermentation in days. The results are shown in Table I and as curve A of the FIGURE.

Also shown in Table I are data for an average of ten runs in which lb. (145 p.p.m.) of ammonia was added on the fourth day after the start of fermentation, which are plotted as curve B of the gure.

5 1 e .mmm t n .D min .IW ren I a e V. a C b ,m m d.d Owem wWumC .mofw .deho md n I anh.,c.1 UCPC E c .10b .Mhe L .nwsm W ce e fbh A o lt ePth. X mb mik o E .nu mhd CilXHWr uwe fm d V. O Ongim d mtmemdnmov fmwnmn Oeoute l a eV1m0.tP tnft1P m... ma eedHS n.. h h e einem u md The fermentation medium was made up in accordance with the teachings of United States Patent No. 2 492 667 D 1, 20 ata for an average of ten runs in which 145 ppm. 'to Raymond L' snfn an? tleoargNB'dSch/gnd of ammonia was added on the fth day after the start Patent No' 2492 3 to o u O0 War an l ay' of fermentation are also shown in Table I and plotted as curve C of the figure.

mond L. Snell. The medium was prepared from Cuban high-test molasses which was defecated,v decationized,

Table I Selected Days after Start of Fermentation 10th 11th 3rd. 4th

ltered and made up to Volume. The initial charge to V the fermenter was as follows:

16,500 gal.

Day Ammonia Added Sugar 22,0S01b. (16.08%). Iron 0.40 ppm.

Potassium acid phosphate 18.75 lb. (137 p.p.m.).

l m. P p 7 8 b. .l 2 11 e d .n O m c m u .1 .m a C t From these data and from a comparison of the curves After the nutrient elements Were added to the deionized plotted in ,the gure it can be Seen that addition of am. medium in the fermenter, the pI-I was adjusted from pI-IV monia on the fourth or the fifth day not only prolongs the rapid rate of citricV acid production andthereby decreases the length of the fermentation cycle but also 1 leads to a higher rate than occurs Without ammonia ad- 1.4-1.6 by addition of ammonia to a pH within the range of pH 2.5-2.8. This required from about 400 parts per 40 million to 700 parts per million of nutrient nitrogen.

The vfermenter was then inoculated with spores of Asperdition. The maximum rate is 'seen to Occur about two gillus niger Strain No. 139 according to standard pracdays after ammonia addition' tice, and the fermentation was allowed to proceed for h d The following example illustrates the improved rates ve days' on t e ff day of fermrentauon Pounns of production of citric'acid achieved through the use of 0f anhydrous 'ammoma (800 Parts Per m11 lion) was ammonium carbonate in the process of this invention.

added. The fermentation'medium Washarvestedl after Y EXAMPLE In 12 days and .the yield of citric acid was found to be 85.2%.

VIt may be seen from this example that a yield ofV over The fermentation mediawere made up according to the 85% was obtained using the process of this invention. procedure of Example I except that the media contained 17.63% reducing sugars, 0.25 parts per million iron, 0.1

Prior Vto use of the improvement of this invention, yields of from about 65% to 75% were considered satisfactory. parts per vmillion ZnJfL and 200 parts per million of nitro- The following example illustrates'the Veffect of nitro- 55 gen as Vammonium carbonate at pH 2.72. The data obgen'addition upon the rate of citric acid production.

tained are shown in Table II..

Tabla '11.

...n 5869710 87598 n. m 0.0.0.00LLLnmn0.0.0.0 U

0 t i .MU 9 000110110110L0 Um i 7720700272 2.07 'Wah ...m 8004234321 049 m0 7 QLLLLLLLLLLLI.

t i tr.

a 5 n0-D 227057 ucm um mo7N342m3202-00 ,ww 6 LLQLLLLLLLLILL r PH 55.5 50 2552. da m mw734M11mhw/2039 dw. 5 LLJLLLLLLLLLL Aa D 2225550755502 .md m ...113842322522549 new 4 LLQLLLLLLLILL oa .I 25 05572552 0M .m 22%32M444l4539 3 LLalLLLLLLLL-lo a R 5575 5 57057 m m A n These data show that the rate of citric acid production is markedly increased by the addition of ammonium carbonate according to the process of this invention. The data for -the run in which 4,000 parts per million of monium salts as a nitrogen containing nutrient effective to stabilize the production of citric acid beyond the point at which said production normally tends to decrease, said addition being made at a time which is no earlier than nitrogen as ammonium carbonate was added to the orig- 5 about one day from the point of maximum rate of citric inal fermentation medium and no nitrogen nutrient was acid production in the absence of said added nutrient. added after the beginning of fermentation show that the 2. In a process for the production of citric acid which addition of even twice as much nitrogen nutrient prior comprises yfermcnting a carbohydrate medium with a to the beginning of fermentation does not result in the citric acid producing strain of a fungus, said carbohyimproved rates of citric acid production which are ob- 10 drate medium containing nutrients including a nitrogen served for additions in accordance with the teachings of nutrient for said fungus, the improvement which comthis invention. prises adding to said fermenting medium a quantity of a The following example illustrates the use of ammonium member selected from the group consisting of ammonia hydroxide in the process of this invention and the remarkand ammonium salts as a nitrogen containing nutrient able results achieved thereby. suflficient to furnish nitrogen in a concentration of from about 50 parts per million to about 5,000 parts per mil- EXAMPLE IV lion, at a time from about the beginning of the third The fermentation media were made up according to day of fermentation to about the end of the seventh day the process of Example I, except that the media contained thereof. 17.06% initial sugar, 0.32 parts per million iron, 0.1 part 3. In a process for the production of citric acid by per milliOIl Cll++, 0-1 Part Per million Zn++, and 540 submerged fermentation of a carbohydrate medium with parts per million of nitrogen as ammonium hydroxide. a citric acid producing strain of Aspergillus niger, said The data obtained are shown in Table III. medium containing nutrients, including a nitrogen nu- T able III Ammonium Rate of Citric Acid Production (g. /100 ml. lday) Hydroxide Added on Selected Days after Start of Fermentation Days p.p.m 3rd 4th 5th 0th l 7th 8th 9th 10th 11th These data ShOW Clearly that the rate of citric acid trient, for said strain of Aspergillus niger, the improve- Production iS 110t Only Stabilized at an increased rate for ment which comprises adding to the fermenting medium an @Xteflded Period 0f time by the addim? 0f ammonifm an amount of a member selected from the group consisthYdfPXld? in accordance with the teaclmgs 0f, 'd ls 11?' 45 ing of ammonia and ammonium salts as a nitrogen con- Vennon but-a1s0 that the Tate of Pfoduciln of mmc acid taining nutrient effective to furnish to said fermenting llatlilta higher level through thls addmon of mtrogen medium a concentration of from about 50 parts per mil- In summary, this invention is directed to the improved l i0 359m 5000 parts Ber inhon of mtmgen Salld process for the production of citric acid by the submerged 50 n no? 31mg made at a point m the feimentatwnpyc e vegetative fermentation of carbohydrates with citric acid Whlch lswhlthu about om? day of the Pomt of mfaxlmum producing Strains of fungi Such as Aspergillus niger rate .of c1tr1c acid producuon 1n the absence of sa1d added wherein a nitrogen nutrient is added to the fermentation numentmedium during fermentation at a time at which the addi- 4- The process of claim 1 wherein the mtrogen c011- tion of nitrogen is effective to increase `and maintain the taining nutrient is an ammonium ion furnishing nutrient. rate of citn'c acid production. As a result, yields obtained 5. The prCeSS 0f Claim 1 Whrein the nitrogen c011- are greatly improved over yields previously attainable by taining nutrient isv ammonia. the use of known submerged fermentation processes and 6- The process of claim 3 wherein the nitrogen confermentation times can be markedly reduced. taining nutrient is ammonium hydroxide.

What iS Claimed iS 60 7. The process of claim 1 wherein the nitrogen con- 1. In a process for the production of citric acid which taining nutrient is ammonium rbonatg comprises subjecting a carbohydrate to fermentation with a citric acid producing strain of a fungus in the presence References Cited in the file of this patent of nutrients for said fungus, said nutrients including a nitrogen nutrient, the improvement which comprises add- UNITED STATES PATENTS ing to the fermentation broth a quantity of a member se- 2,394,031 Waksmall et al Feb 5, 1946 lected from the group consisting of ammonia and am- 2,910,409 Fried et al OC- 27, 1959 

1. IN A PROCESS FOR THE PRODUCTION OF CITRIC ACID WHICH COMPRISES SUBJECTING A CARBONHYDRATE TO FERMENTATION WITH A CITRIC ACID PRODUCING STRAIN OF A FUNGUS IN THE PRESENCE OF NUTRIENTS FOR SAID FUNGUS, SAID NUTRIENTS INCLUDING A NITROGEN NUTRIENT, THE IMPROVEMENT WHICH COMPRISES ADDING TO THE FERMENTATION BROTH A QUANTITY OF A MEMBER SELECTED FROM THE GROUP CONSISTING OF AMMONIA AND AMMONIUM SALTS AS A NITROGEN CONTAINING NUTIENT EFFECTIVE 